This invention relates to a method of demarcating a two-dimensional distribution in which, when a specimen containing two or more types of particle groups is measured by a particle analyzer capable of measuring two analytical parameters regarding various particles of interest, one particle group is differentiated from other particle groups by drawing a discriminant on a plot of a two-dimensional distribution in which the two analytical parameters are adopted as the two axes.
In order to demarcate one particle group from another particle group in particle analysis, a common practice in the art is to draw a plot of a two-dimensional distribution regarding the particles of interest and draw a discriminant within the plot of the distribution. For example, the specification of U.S. Pat. No. 4,325,706 describes a method of measuring a blood sample, which is fluorescently dyed with acridene orange, by means of a flow cytometer, and differentiating the particle groups in the blood sample by drawing a discriminant within a plot of a two-dimensional distribution prepared from signals indicative of fluorescence and scattering detected from individual particles. FIG. 9 is a view of FIG. 8B of the drawings in the above-mentioned specification. The horizontal axis represents the signal strength of red fluorescence detected from the particles, and the vertical axis represents the signal strength of scattered light detected from the particles. A red blood cell (RBC) particle group and a reticulocyte (RETICS) particle group in the center of the view are demarcated from a platelet (PLT) particle group by a diagonally extending linear discriminant.
In a case where particle groups on a plot of a two-dimensional distribution are differentiated by a linear discriminant as set forth above, the manner in which the discriminant is drawn is simple and no particular problems are encountered. In actual practice, however, there are many instances in which it is necessary to draw a more complicated discriminant. Since the acridine orange used in the aforesaid U.S. patent exhibits strong background fluorescence due to the dye solution itself, the dye tends to produce an error in the measurement of the intensity of fluorescence ascribable to the blood cells. Thus, it has been found that this dye is difficult to actually use. These findings are set forth in the specification of Japanese Patent Application Laid-Open (KOKAI) No. 61-280565. Accordingly, the same publication discloses use of a more practical dye (auramine 0) instead of acridine orange as a reagent for measuring reticulocytes. When auromine 0 is used, however, the red blood cell particle group and reticulocyte particle group cannot be demarcated from the platelet particle group by a simple linear discriminant, as shown in FIG. 1. Here the horizontal axis represents the relative intensity of fluorescence and the vertical axis the relative intensity of forward-scattered light. Each point in FIG. 1 corresponds to an individual particle, A represents the red cell particle group, B the reticulocyte particle group and C the platelet particle group. The curved line in FIG. 1 is a discriminant which demarcates the red cell particle group and reticulocyte particle group from the platelet particle group. The vertically extending straight line in the plot of FIG. 1 does not have a direct bearing upon the present invention but is a discriminant distinguishing the red blood cell group from the reticulocyte particle group. This line is shown for reference purposes only.
The characters and lines in FIGS. 2 through 6 have the same meansings. FIG. 1 is a plot of results obtained when measuring the blood sample of a healthy individual. When a specimen having an idiosyncrasy is measured, however, the distribution of the platelet particle group (C) exhibits a greater spread toward the upper right in comparison with the healthy individual, as shown in FIG. 2. As a result, the positions of the discriminants in FIGS. 1 and 2 clearly differ. This means that it is necessary to find an optimum discriminant for each and every sample in order to accurately quantify the number of platelet and/or reticulocyte particles.